Pieternel M. Rademaker

Estimation of classical pathway of mouse complement activity by use of sensitized rabbit erythrocytes.

A simple photometric assay was devised for determining classical complement pathway activity in mouse serum using sensitized rabbit erythrocytes as target cells. These cells appeared more sensitive to lysis by mouse complement than sensitized mouse and sheep erythrocytes, most probably by their ability to escape the C3b inactivator system. Advantages of the assay over other techniques are the high sensitivity and the avoidance of the use of radioisotopes. With this test it is possible to get more insight in the complement system of an animal species that has been most widely in use in immunological research.

Study of the optimal reaction conditions for assay of the mouse alternative complement pathway

The optimal reaction conditions for hemolytic assay of alternative complement pathway activity in mouse serum were investigated. A microtiter system was used, in which a number of 7.5 X 10(6) rabbit erythrocytes per test well appeared to be optimal. Rabbit erythrocytes were superior as target cells over erythrocytes from a number of other animal species. The optimal conditions were as follows: an incubation temperature of 39 degrees C, an ionic strength of about 200 mM, and a magnesium concentration of 2.5 mM. Incubation during 60 min was not sufficient for an end-point titration. Addition of 1 mg of zymosan A per test well, however, enhanced and accelerated the hemolytic activity of mouse serum via the alternative pathway resulting in a maximum value after 45 min. This, most probably, proceeded by a mechanism involving the formation of a zymosan-C5-convertase and bystander lysis of the target cells. In contrast to the normal alternative pathway assay the zymosan-potentiated test did, most probably, not involve natural antibodies. Cobra venom factor was more efficient in enhancing the sensitivity of the assay for the mouse alternative complement pathway than zymosan. This makes this factor very useful for testing C-poor body fluids.

Effects of immunological adjuvants on the mouse complement system. I. The inability of the polyanion heparin to act as an adjuvant is paralleled by inefficient alternative complement pathway inhibition.

Interference with hemolytic complement activity by polyanionic substances was studied in relation to the ability of these compounds to act as an adjuvant for a dead listeria vaccine. Heparin appeared a poor inhibitor of the mouse alternative pathway not only in contrast to its effects on the mouse classical and the human classical and alternative pathways, but also when compared to two polyanions with known adjuvant activity: dextran sulfate and suramin. For the three polyanions mentioned a correlation between adjuvant activity and mouse alternative pathway inhibition was observed. These findings suggest a possible causal relationship between interference with alternative complement pathway activation and adjuvant activity.

Involvement of membrane-associated sialic acid in the resistance of sheep erythrocytes to lysis by mouse complement

The sensitivity of the murine complement system to regulation by membrane-associated sialic acid was investigated. Therefore the C-mediated lysis of sialic acid-rich sheep erythrocytes, sialic acid-poor rabbit erythrocytes and enzymatically desialylated sheep erythrocytes was studied. Mouse complement differed from human and guinea pig complement in that besides the alternative, also the classical pathway appeared sensitive to regulation by surface sialic acid. The possible reaction mechanisms behind the selective sensitivity of the murine classical pathway to membrane-linked sialic acid are discussed and will be subject of further study.

A study of the sensitivity of erythrocytes to lysis by heterologous sera via the alternative complement pathway

In order to get insight in the distribution of alternative complement pathway activities as detected by lysis of xenogeneic erythrocytes in the presence of magnesium and ethyleneglycol-bis-(2-aminoethyl)-tetra-acetic acid (EGTA) over the species, the 156 heterologous combinations of erythrocytes and sera out of thirteen animal species were tested. An order could be noticed in the species with respect to serum complement activity tending to negative correlation with the sensitivity of the corresponding erythrocytes to lysis by heterologous sera. So far, the most sensitive erythrocyte for each individual serum must be considered to be the target cell of choice for developing assays for alternative complement pathway activity in the serum involved. In this series of animals only for rabbit serum no sensitive target cell was found. The order observed, in connection with the failing lysis of erythrocytes by homologous sera, suggests further that in restriction of heterologous hemolysis in general one erythrocyte-associated, species-nonspecific regulatory principle may be involved, whereas in homologous restriction, most probably, also species-specific factors play a role.

Histamine 2-receptor-mediated immunomodulation in the mouse. II. Immunomodulation by the H2 antagonist metiamide

Modulation of delayed hypersensitivity and antibody formation to sheep red cells by metiamide were studied in the mouse system. Depending on the time and dose of antigen and metiamide administration suppression or enhancement of the delayed hypersensitivity response was observed. The effects in this system did not differ from those reported for the H2 agonist tolazoline, which were most probably mediated by suppressor cells. As far as the humoral response was concerned metiamide tended to stimulate the IgM response. Optimal stimulation was reached if 50 mg metiamide/kg was administered 3 days before immunization with 2 x 10(8) SRBC. Under all conditions tested the IgG response was unaffected. These results suggest antagonistic effects of metiamide for tolazoline and adduce further evidence for the presence of H2 receptors on B cells. The IgM production per plasmacell was enhanced suggesting different H2 receptors to be involved in differentiative and proliferative B cell responses. The possible consequences of H2 antagonist application in human therapy are discussed.

House dust allergen activates the classical complement pathway in mouse serum

A house dust fraction was tested for complement activation in mouse serum using a microtitre complement fixation assay. It was observed that the preparation was a potent activator of the classical, but not of the alternative pathway suggesting an analogy with the complement activation in human serum. The activation showed similarity with that by classical complement activators such as aggregated IgG, DNA, lipopolysaccharide (LPS), but some discrepancy with mite allergen was observed. The contamination of the preparation with LPS was negligible and could not account for the anticomplementary effect. The role of DNA fragments in the activation of mouse complement by the house dust fraction is discussed. Our results suggest that the mouse is suited to study the role of complement activation by house dust constituents in the induction of the IgE response.

Surface-associated sialic acid is an immunological adjuvant

The influence of neuraminidase on the immunogenicity of heterologous erythrocytes as determined by serum haemagglutination titres was investigated in mice. For this study sheep and rabbit erythrocytes were selected because of their high and low N-acetylneuraminic (sialic) acid content, respectively. Preincubation with neuraminidase resulted in a ten-fold reduction of the immunogenicity of sheep erythrocytes (ShE). By contrast, the immune response to rabbit erythrocytes appeared to be resistant to sialidase treatment. Addition of the extrinsic adjuvant dimethyldioctadecylammonium bromide largely restored the immunogenicity of neuraminidase-treated ShE, but did not change the response to control-treated ShE. The maximal antibody level induced by neuraminidase-treated ShE was lower than that provoked by control ShE. These results suggest that sialic acid is both an intrinsic immunological adjuvant and an antigenic determinant of ShE. The adjuvant effect of sialic acid does not depend on complement component C3 as judged by the response of cobra venom factor-pretreated animals. In genetically C5-deficient and in nude mice, however, sialic acid showed diminished and absent adjuvant activity, respectively.

Restricted rosette formation with an EAC reagent prepared with serum of C3H/HeJ mice

The EAC generating capacities of fresh and stored BALB/c, Swiss, C3HeB/FeJ, and C3H/HeJ mouse sera were investigated in a rosette test using spleen cells of each of the serum donors as indicator cells. With BALB/c spleen cells a decreased ability of both stored and fresh C3H/HeJ serum to generate functional EAC could be registered, whereas with C3HeB/FeJ spleen cells such was the case only with stored serum. Swiss spleen cells did not discriminate between the capacities of the four sera and with C3H spleen cells both C3H sera tended to be less effective than BALB/c and Swiss serum. This indicates that C3H/HeJ serum is able to generate EAC but with a somewhat different disposition of cell-bound C molecules. Our results suggest that the “defect” in C3H/HeJ serum is localized in C4, C3, or rather in some C-associated inhibiting system as C4-binding protein or the C3b inactivating system. A possible relationship of the “defective” serum factor and the defective Lps gene product is suggested.

House dust extracts contain potent immunological adjuvants

A crude aqueous extract of house dust and two house dust subfractions were tested for adjuvant activity in a sensitivity assay performed in mice. Evidence is presented that house dust contains at least two potent immunological adjuvants. One of these, present in both subfractions, was probably endotoxin and acted in a complement-independent way. The immunostimulatory effect of the other adjuvant was abrogated by prior complement depletion of the animals. This apparently complement-dependent adjuvant needs further identification.